The slow rate of membrane-protein structure determination represents a significant bottleneck for both basic and applied bioscience discovery, thus a tremendous need exists for innovative methodological breakthroughs. We propose to revolutionize NMR structure determination of ?-helical, polytopic membrane proteins, with specific focus on those from mitochondria, through the employment of DNA-nanostructure-based alignment media. Recently, we developed a detergent-resistant liquid crystal of six-helix-bundle DNA-nanotubes that shows great promise as a robust tool for weak alignment of membrane proteins. Weak alignment enables measurement of global angular restraints that can serve as the primary source of structural information in studies of ?-helical membrane proteins, where a sufficient number of distance restraints can be impossible to obtain; thus the effective size limit can be raised from 15 kDa to over 40 kDa. Realizing the potential of this technology to make feasible the NMR structure determination of a wide range of membrane proteins will require the development of additional DNA-based alignment tools that improve compatibility with positively- charged proteins and that enable measurement of additional structural restraints. Towards these two ends, we will build and characterize the alignment capabilities of novel DNA nanostructures that either are longer, are coated with polyethylene glycol, or have helical axes perpendicular to the long axis of the alignment particles. We also will apply our DNA nanotools towards the NMR structure determination of peripheral benzodiazepine receptor, a 18 kDa polytopic ?-helical, polytopic membrane protein that is involved in the steroidogenesis- limiting import of cholesterol across the outer mitochondrial membrane. This opportunity to advance membrane-protein structural biology arises from recognition of the need for custom-shaped, detergent- resistant materials matched with the unique expertise of the PI and his laboratory to self-assemble large, arbitrary 3D shapes from DNA.